Three nitric oxide synthase enzymes (NOS) evolved to function in human health and disease. We wish to define the mechanisms and the protein structural features that regulate NOS catalysis. The NOS flavoprotein domain transfers NADPH electrons to a heme in the oxygenase domain and this enables NO synthesis. Catalytic regulation is complex because NO binds to the NOS heme before it exits the enzyme. We developed a global kinetic model that incorporates these and other facets of catalysis. The global model posits that the unique catalytic profiles of the three NOS are due to differences in three kinetic parameters: Ferric heme reduction (kr), ferric heme-NO dissociation (kd), and oxidation of the ferrous heme-NO complex (kox). The mechanisms and structural basis for controlling NOS electron transfer and for setting the kr, kd, and kox values are still unclear. Our Aims propose biochemical, kinetic, molecular biological, and biophysical studies to tackle these issues, in order to provide a molecular-level understanding of NOS control mechanisms. Aim 1. What regulates electron transfer and heme reduction (kr) in NOS? Electron transfer by the FMN module is a central feature of NOS catalysis. We will investigate how partner subdomains, FMN redox status, &calmodulin regulate the conformational equilibrium of the FMN module and its interactions with electron acceptors, how inter-domain surface charge interactions regulate FMN electron transfer and NOS catalysis, and how a specific FMN-protein interaction and the composition of two connecting hinge elements govern FMN module electron transfer in NOS. Aim 2. What regulates reaction of the ferrous heme-NO complex with O2 (kox)? The kox reaction is key because it determines NOS catalytic behavior and generates an N-oxide product that is distinct from NO. We will investigate how kox may be regulated by the NOS heme midpoint potential, protein structural features that control O2 access to the heme, and interactions between the FMN and NOSoxy subdomains, and will study the kox mechanism and regulation in several related heme-thiolate enzymes. Aim 3. How do NOS enzymes regulate their NO release (kd)? Factors that control ferric heme-NO dissociation (kd) are particularly important for NOS because its newly-generated NO molecules coordinate to the ferric heme before leaving the enzyme. We will investigate how the NO kd is controlled by NOS dimeric structure, substrate binding &structure, and the size of the heme pocket opening. Aim 4. Generate NOS with novel catalytic behaviors through protein engineering. Within the context of Aims 1-3 we will create NOS variants that possess non- native combinations of kr, kox and kd. These will test the global model and our understanding of kinetic control mechanisms in NOS, will reveal if NOS enzyme function can change when the kinetic parameters exceed their natural ranges, and may generate useful super-NOS variants for NO delivery therapy. Relevance: By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide. PUBLIC HEALTH RELEVANCE: Three nitric oxide synthase enzymes function broadly in human health and disease. We wish to determine how the protein components of these enzymes regulate their nitric oxide production. By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.